The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Summary

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

Abstract

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry will help to ensure LAB viability and functionality for many applications.

Introduction

Lactic acid bacteria (LAB) are a group of uniquely diverse bacteria that have industrial potential. Strains belonging to Lactobacillus delbreuckii subsp. bulgaricus and Streptococcus thermophilus are mostly used as dairy starter cultures for fermented dairy food products such as yogurt¹. Selected LAB strains are also classified as probiotics as they confer health benefits to humans when dosages are adequately administered². Lactic acid bacteria are also gram-positive, non-spore-forming, non-respiring but aerotolerant microorganisms that are generally characterized by the production of lactic acid as a key fermentation product. LAB also synthesizes essential metabolites, for example, organic acids, bacteriocins, and other antimicrobial compounds³ that can inhibit a broad spectrum of foodborne pathogens⁴. Lactic acid, a major end product of carbohydrate catabolism and a by-product of LAB fermentation, is an organic metabolite that possesses antimicrobial properties and is potentially useful for food biopreservation applications^3,5,6. Furthermore, the organic acids produced by LAB impart the flavor, texture, and aroma of foods, thus consequently enhancing their overall organoleptic properties^5,6. The distinct nutritional requirements of LAB coupled with their ubiquitous nature, ultimately enable the bacteria to easily thrive in different environments such as dairy-based foods, fermented foods, vegetables as well as in the human gut⁷.

There is a growing demand for starter cultures from LAB for yogurt production and many diverse dairy applications^8,9, hence critical attention and established scientific techniques should be adhered to, in LAB strains cultivation, as well as in the activation of both lyophilized and isolated strains as this activity is vital for enhanced fermentation performance. The Food Microbiology and Biotechnology laboratory, therefore, actively engages in suitable technology development geared toward the activation, superior growth, and fermentation characteristic of LAB strains isolated from fermented dairy products as well as from industrial starter cultures employed for yogurt production. Furthermore, it is noteworthy that LAB culture strains industrially produced undergo preservative activities such as freeze-drying and frozen storage, causing cell stress and injury, as a result of the cold shock process they are subjected to¹⁰. In limiting, the viability challenges and improving the functionality of LAB strains obtained from either isolated food products or freeze-dried products, it is important to properly activate these cultures as a form of quality control to enhance their fermentative characteristic⁸. In this study, the objective was to develop an in-house quality control protocol for the activation and growth of L. delbrueckii subsp. bulgaricus culture strains that ultimately promoted viable LAB growth, as well as enhanced the fermentation performance and the metabolic functionality of LAB strains. This protocol could ultimately be adapted (using optimal growth media and appropriate culturing conditions) for the cultivation of other LAB strains for fermentation research, as well as for industrial purposes or bioprocessing operations. This LAB activation and quality control protocol will therefore ensure superior viable dairy starter cultures are obtained and potentially functional for diverse applications in the global dairy and food industry.

Protocol

1. General materials and methods

1. Source of Lactobacillus delbrueckii subsp. bulgaricus
   1. Obtain L. bulgaricus strains from reliable sources.
      NOTE: In this study, a total of five (5) L. bulgaricus strains were used in the quality control study (Table 1). Two strains of freeze-dried L. bulgaricus for the industrial production of fermented milk products were provided by Dr. Albert Krastanov, Department of Biotechnology at the University of Food Technologies, Plovdiv, Bulgaria. Two strains isolated from commercial yogurt products available in the US market were obtained from the -80 °C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&T State University and one freeze-dried strain was supplied by a vendor C. All LAB strains were kept at -80 °C until further use. Another bacterial strain (Limosilactobacillus reuteri) was obtained from the -80 °C stock of the Food Microbiology and Biotechnology laboratory at North Carolina A&T State University was also evaluated.
2. Standard L. MRS fermentation broth medium
   1. Prepare deMan, Rogosa Sharpe (MRS) medium by completely dissolving 55 g of MRS, and 0.5 g of L-Cysteine in 1 L of deionized water (DW).
   2. Dispense 7 mL and 2 mL of the prepared MRS solution into test tubes, respectively, autoclave at 121 °C for 15 min, and then cool at room temperature (RT).
3. MRS agar medium
   1. Prepare MRS agar medium by completely dissolving 55 g of MRS and 0.5 g of L-Cysteine in 1 L of DW. Add agar powder (15 g), sterilize the agar medium at 121 °C for 15 min, and then cool it in a water bath.
   2. Pour all freshly-prepared media into sterile Petri dishes and store them at 4 °C until further needed.
4. Modified reinforced clostridial medium-pyruvate (mRCM-PYR)
   1. Optimize a reinforced clostridial medium according to Oyeniran et al.¹³ and Nwamaioha and Ibrahim¹⁸, for selectivity and accurate enumeration of L. bulgaricus by dissolving 10 g of peptone #3, 10 g of beef extract, 5 g of yeast extract, 5 g of sodium chloride, 3 g of sodium acetate, 2 g of potassium phosphate dibasic_, 0.1 g of uracil, 0.25 g of calcium chloride, 5 g of dextrose, 5 g of fructose, 10 g of maltose, 2 g of sodium pyruvate, 0.2% Tween 80, and 0.5 g of L- Cysteine in 1 L of DW.
   2. Adjust the final pH (6.0 ± 0.2) of the solution using 6 N HCl before the addition of 0.008% aniline blue and 15 g of agar. Autoclave the medium at 121 °C for 15 min, cool in a water bath, and pour into sterile Petri dishes. Store all freshly-prepared media in sterile Petri dishes at 4 °C until needed.
5. Glycerol stock of LAB cultures
   1. Prepare glycerol stocks (50% glycerol) by diluting 100% glycerol in the same volume of DW and sterilize at 100 °C for 30 min using a dry sterilization cycle. Cool the glycerol stocks to RT and aseptically store them for further use.
   2. Use bacterial growth (a single colony of L. bulgaricus obtained using the developed QC protocol) from overnight cultures.
   3. Pipette 500 µL of the overnight cultures into 50% glycerol in a 2 mL centrifuge tube and gently mix them together. Freeze and store the glycerol stock containing the LAB cultures at an ultra-low temperature of -80 °C until needed.
      ​NOTE: A graphical scheme of the protocol for the quality control and activation of LAB cultures is shown in Figure 1.

No	Product Code	Sample	Source	Bacterial Composition as labeled1
1	S9	Pure Industrial Strain	Bulgaria	Lb. bulgaricus
2	LB6	Pure Industrial Strain	Bulgaria	Lb. bulgaricus,
3	ATCC 11842	Pure Industrial Strain	ATCC	Lb. bulgaricus
4	DAW	Yogurt	USA	Lb. bulgaricus, other live culture
5	E22	Yogurt	USA	Lb. bulgaricus, other live culture
6	Reuteri	Yogurt	USA	Limosilactobacillus reuteri
1Lb. =Lactobacillus

Table 1: Probiotic strains. The table lists the probiotic strains used in this study.

2. Protocol for the activation and quality control of LAB cultures

1. Take the prepared glycerol stock of LAB strains (in 2 mL centrifuge tubes) from the -80 °C ultra-low freezer, and do not allow them to thaw before use.
2. Clean and disinfect the opening of the centrifuge tubes with 70% alcohol, and gently vortex before use.
3. Pipette about 250 µL (0.25 mL) of the stock LAB culture from the centrifuge tubes into fresh 2 mL MRS test tubes.
4. Gently vortex, parafilm the test tubes, and anaerobically incubate them overnight at 42 °C for 12-16 h.
5. Take about 500 µL (0.5 mL) from the overnight grown cultures from the 2 mL MRS test tubes into fresh 7 mL MRS test tubes, vortex, and anaerobically incubate them overnight at 42 °C for 12-16 h.
6. Assess the microbial growth by measuring the optical density (OD) or growth of the cultures at 610 nm with a UV- visible spectrophotometer and record acceptable results between 0.7 and 0.9.
7. Streak the overnight cultures from the 7 mL MRS tubes onto MRS and MRCM-PYR agar plates and incubate them anaerobically for 72 h at 42 °C.
8. Pick isolated colonies from the agar plates, transfer them into fresh 7 mL MRS test tubes, gently vortex, and anaerobically incubate them overnight at 42 °C for 12-16 h.
9. Store the agar plates containing the isolated strains at 4 °C in the refrigerator for a week.
10. Measure and confirm the OD (between 0.7 and 0.9) from the 7 mL MRS test tubes of the LAB cultures isolated from the streaked plates at 610 nm and use them as working cultures for all related experiments.
11. Perform ten-fold dilutions (serially dilute) of the grown LAB cultures from the final 7 mL MRS test tubes using 9 mL of peptone water (a physiological buffer) to obtain a 1:10 ratio.
12. Finally, take about 250 µL (0.25 mL) from appropriate serial dilutions for all fermentation experiments.
13. Activate the broth containing strains (250 µL) from step 2.12 by transferring them into fresh 7 mL MRS broth and incubating them anaerobically at 42 °C for 16 h.
14. Continue by repeating steps 2.6-2.12 to ensure viable and superior cell growth from LAB cultures.
    NOTE: All L. bulgaricus strains were activated in the freshly prepared MRS broth and were then incubated anaerobically at 42 °C for 16 h in order to reach an optical density (OD_{610 nm}) of bacterial growth between 0.7 and 0.9. Bacterial growth was measured with a UV-visible spectrophotometer at 610 nm. The pH values of the overnight cultures were in the range of 3.5 to 5.3 as a result of the production of lactic acid, an organic end product of LAB fermentation. Safety procedures such as proper airflow circulation in the biosafety hood and avoiding burns during the use of the bunsen burner were adhered to and observed.

Figure 1: A graphical scheme of the protocol for the activation of lactic acid bacteria (LAB) cultures. The scheme provides details and the basic instruments required for the handling and activation of LAB culture strains. Please click here to view a larger version of this figure.

Results

Cell growth of the evaluated LAB strains cultivated with the quality control protocol was significantly different (P < 0.05) than the strains cultivated without this standard protocol. The QC protocol for both L. bulgaricus and L. reuteri employed a multi-subculturing approach (subculturing three times before streaking on agar plates), whereas the control procedure had subculturing done only once with all other conditions kept constant. The colony growth was also higher and well defined on the growth...

Discussion

The results of all strains evaluated with the quality control protocol and without the use of the protocol were the same, and as such, results linked to only strains (S9, and LB6) were presented. The activated LAB strains had superior cell growth that was characterized by a high intensity of cell biomass, therefore, causing a turbid appearance of the MRS fermentative broth in the test tube¹¹. The observed cell growth after culture activation was evident between 12 h and 16 h at an anaerobic fermen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aniline Blue	Thermo Scientific	R21526	25 g
Beef extract	Research Products International	50-197-7509	500 g
Yeast extract	Fisher Scientific	BP1422-500	500 g
Calcium Chloride dihydrate	Fisher Scientific	C79-500	500 g
Dextrose Anhydrous	Fisher Scientific	BP350500	500 g
D-Fructose	ACROS Organics	AC161355000	500 g
Difco agar powder	Difco	DF0812-07-1	2 kg
TPY agar	Difco	211921	500 g
Eppendorf microcentrifuge tube (Snap-Cap Microcentrifuge Safe-Lock)	Fisher Scientific	05-402-12	2 mL
Glycerol	Thermo Scientific	PI17904	500 mL
Infrared CO2 Incubator	Forma Scientific
Lactobacillus delbrueckii subsp. bulgaricus	American Type Culture Collection (ATCC)	ATCC 11842
Lactobacillus delbrueckii subsp. bulgaricus	Bulgaria	S9
Lactobacillus delbrueckii subsp. bulgaricus	Bulgaria	LB6
Lactobacillus delbrueckii subsp. bulgaricus	Food Microbiology and Biotechnology Laboratory (NCATSU)	DAW
Lactobacillus delbrueckii subsp. bulgaricus	Food Microbiology and Biotechnology Laboratory (NCATSU)	E22
Limosilactobacillus reuteri	Biogai, Raleigh / Food Microbiology and Biotechnology Laboratory (NCATSU)	RD2
L-Cysteine hydrochloride monohydrate	Sigma-Aldrich	C6852-25G	25 g
Maltose monohydrate	Fisher Scientific	M75-100	100 g
MRS broth	Neogen	50-201-5691	5 kg
Peptone No. 3	Hach	50-199-6719	500 g
Potassium phosphate dibasic (K2HPO4)	Research Products International	50-712-761	500 g
Sodium acetate trihydrate	Fisher Scientific	S220-1	1 kg
Sodium chloride	Fisher Scientific	BP358-1	1 kg
Sodium pyruvate	Fisher Scientific	BP356-100	100 g
Test Tubes with Rubber-Lined Screw Caps	Fisher Scientific	FB70125150	25 x 150 mm
Tween 80	Fisher Scientific	T164-500	500 mL
Ultra low freezer	So-Low
Uracil	ACROS Organics	AC157301000	100 g
UV- visible spectrophotometer	Thermo Fisher Scientific	Evolution 201
Vortex Genie 2	Fisher Scientific
Yeast extract	Fisher Scientific	BP1422-500	500 g
Ethanol	Fisher Scientific	T08204K7	4 L
Hydrochloric Acid (6N (Certified), Fisher Chemical)	Fisher Scientific	SA56-500	500 mL